In the examination of histological sections of liver, it is important to distinguish bile pigments from lipofuscin.
Both bile pigments and lipofuscin are appeared in yellow-brown colour in H and E-stained sections.
Moreover, green colour biliverdin are also masked by eosin when stained with H and E stain.
For the identification of bile pigments, Fouchet’s staining technique is most commonly used.
By the oxidative action of ferric chloride Fouchet’s reagent converts bile pigments to green colour biliverdin and blue colour cholecyanin.
This occurs in the presence of trichloroacetic acid.
Van Gieson’s solution is used as the counter stain.
When counter stained with van Gieson’s solution, green colour is accentuated.
Depending on the concentration of bile pigments the colour of the pigment range from olive green to emerald green.
For the preparation of Fouchet’s solution, mix 36 ml of 25% aqueous trichloroacetic acid with 4 ml of 10% aqueous ferric chloride.
This Fouchet’s solution should be prepared freshly for the test.
100 mg of acid fuchsin is dissolved in 100 ml of saturated aqueous picric acid For the preparation of Van Gieson stain.
- First take the test tissue section and the control section down to the water.
- Then stain with the freshly prepared Fouchet’s solution for 10-12 minutes.
- Then by using running tap water wash the sections about 1 minute.
- After that rinse in distilled water.
- Next by using van Gieson solution counterstain the sections for 2 minutes.
- Finally dehydrate, clear and mount the sections with a synthetic resin.
Bile pigments are visible as emerald to blue-green colour. Muscles stain in yellow colour and collagen stain in red coour.
Any fixative can be used for the fixation of tissues used for this technique.
Two control slides should be used for this.
One section is stained with Fouchet’s reagent and van Gieson solution.
The other section is stained with Fouchet’s reagent alone.
Freshly prepared reagents give more reliable results.
Furthermore, Bile can be present outside the liver such as the Aschoff-Rokitansky sinuses or in hemorrhagic and infarcted areas.
In that type of situations, no colour change can be seen with Fouchet’s method.
For this type of bile pigments there is another test called Gmelin or Stein technique.
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